Study On Inhibition Effect Of IFITM2 On Pseudorabies Virus Replication And The Relevant Antiviral Mechanism

Pseudorabies virus(PRV)is one of the most important viral infectious factors that cause reproductive disorder in sows.PRV can infect pigs of all ages.The occurrence of abortion,stillbirth,mummified foetus and other phenomena bring great economic loss to the pig industry.We detected PRV g E gene in 89 foetus,results found that the infection rate was up to 12.4%,and it had a high infection in spring and winter,which indicated that PRV was closely related to abortion.However,the mechanism of PRV infection and abortion is still unclear,so it is necessary to study the mechanism of PRV causing reproductive disorder in sows,to reduce the mortality of foetus and piglets,and to ensure the economic benefit of raising pigs.Interferon-induced transmembrane proteins(IFITMs)are small molecules with biological activity,which play an important role in host resistance to pathogen infection.In recent years,many studies have proved that human and mouse IFITMs can resist the replication of many kinds of RNA viruses,but there are few studies on the inhibition role of IFITM proteins from swine.Therefore,a series of studies have been carried out to explore the role of porcine IFITM2 protein in the process of PRV infection and the relevant antiviral mechanism.The results are listed as follows:(1)Effect of IFITM2 protein on PRV replication.IFN-α2a stimulation could effectively induce the expression of swine IFITM1,IFITM2 and IFITM3 protein in PK15 cells.Then successfully constructed expression vector p CMV-Myc-IFITMs.The expression of IFITMs in PK15 cells was confirmed by western blotting.Results showed that IFITMs were successfully expressed in PK15 cells,which laid a foundation for further antiviral research.Transient overexpression of IFITM protein cell lines were used to valid the effect of IFITM1,IFITM2 and IFITM3 on PRV infection,results indicated that IFITM proteins significantly inhibited PRV replication.In the study of antiviral effect of IFN,RNA interference(RNAi)was used to down-regulate IFN-α2a-induced IFITM2 protein expression.Then followed by PRV infection.It was found that virus replication increased during IFITM2 expression down-regulated,that is,the antiviral effect of IFN was weakened.These results suggest that swine IFITM2 protein can effectively inhibit PRV infection in host cells and is associated with the antiviral activity of IFN.(2)The mechanism of IFITM2 inhibited PRV replication.In order to further explore the mechanism of inhibition of PRV replication by IFITMs,the effect of IFITMs on PRV adsorption and entry were studied.Results found that IFITM1,IFITM2 and IFITM3 could significantly inhibit the entry of PRV.IFITM2 also inhibited the adsorption of the virus.The down-regulated expression of IFITM2 by RNAi had no effect on cell viability,but the absorption and entry of virus were enhanced obviously,which suggested that IFITM2 protein played a certain role in maintaining the antiviral activity of cells during virus infection.The decrease of PRV infection in cells treated with MβCDX and PF429242 suggests that PRV infection is related to cholesterol pathway.So overexpression IFITM2 cells were treated with PF429242,then verified the inhibition of IFITM2 on the virus.The inhibition of IFITM2 on virus adsorption,entry and replication was significantly weakened,suggesting that IFITM2 inhibits the proliferation of PRV through cholesterol pathway.(3)Effect of PRV and its viral protein US3 on type I IFN signaling pathway.PRV had no effect on the stable expression of IFITM2 in PK-IFITM2 cell lines,but inhibited IFN-α2a induced IFITM2 expression,suggesting that PRV might interfere with type I IFN signaling pathway.PK15 cells transfected with poly(I:C)and followed by PRV infection.Cellular total RNA was extracted then subjected to RT-q PCR.Results indicated that PRV could inhibit the transcription of IFN-β induced by poly(I:C).Further study found that viral proteins ICP0,UL24,UL26,UL41 and US3 could significantly inhibit the transactivation of IFN-β,and the inhibition effect of US3 was the most significant.As PRV infection could be recognized by c GAS-STING signaling pathway-associated proteins.Then we examined the effect of US3 on the expression of c GAS,STING,TBK1 and IRF3,it was found that US3 could inhibit the m RNA and protein expression of c GAS,STING and IRF3.(4)Mechanism of US3 protein inhibiting type I IFN signaling pathway.By transient overexpression of US3 protein,it was found that US3 inhibited the IFN-β activation mediated by c GAS-STING,TBK1 and IRF3.These result suggest that US3 targets IRF3 or downstream of IFR3 to antagonize IFN-β activation.To further analyze how US3 inhibits the activation of IFN-β,Co-IP and nuclear-cytoplasmic separation experiments were did.Results showed there was directly interaction between US3 and IRF3,and US3 inhibited the process of IRF3 nucleus translocation.IRF3 nuclear translocation needs to interact with KPNA3 and KPNA4,firstly verified whether US3 affected the expression of importin proteins and found that US3 had no effect on the expression of KPNA3 and KPNA4.Co-IP experiments confirmed the interaction between IRF3 and KPNA3 and KPNA4,but the interaction was weakened in the presence of US3.More interestingly,we found there was obviously interaction between US3 and KPNA4.These results confirm the hypothesis that US3 suppresses the activation of IFN-β and the expression of a series of ISGs downstream of IRF3 by combining with KPNA4 to inhibit IRF3 nucleus translocation.In conclusion,this study demonstrated that swine IFITM1,IFITM2 and IFITM3 proteins were found to play an important role in host resistance to PRV infection.IFITM2 inhibited PRV entry and replication and its inhibition role via cholesterol pathway.In addition,viral protein US3 inhibited c GAS,STING and IRF3 mediated IFN-β activation via degaration c GAS,STING and IRF3 expression.US3 competed with IRF3 to combine with KPNA4,then inhibited IRF3 nuclear translocation and type I IFN expression.US3 antagonized host antiviral immune response by inhibiting ISGs m RNA expression.These results may shed light on the potential application of IFITM2 protein as a antiviral drug and provide new scientific information for elucidating the interaction between PRV and the host innate immune response.